Functionalized eyewear device for detecting biomarker in tears

ABSTRACT

Disclosed herein is a functionalized eyewear device that is adapted for collecting and analyzing disease biomarkers. Specifically, exemplified is a contact lens that has aptamer molecules associated therewith for binding to a specific biomarker (or biomarkers). The eyewear device is useful for detecting and diagnosing medical conditions.

BACKGROUND

There is an urgent need to establish noninvasive biomarkers which enableearly detection of a range of diseases such as cancers andneurodegenerative disorders. Recent advances in the field of “omics”have facilitated identification of novel biomarkers from variousbiospecimens. This is well reflected in the recent global biomarkersmarket research anticipating growth from $29.3 billion in 2013 to $53.6billion in 2018, a CAGR of 12.8% (by PRNewswire).

Recent studies have demonstrated that the tear fluid is a uniquebiospecimen containing mucins, lipids, more than 500 proteins, peptides,cytokines and neurotransmitters. In particular, the scarcity of albuminand immunoglobulins in tears while abundant in serum makes analysisstraightforward. Changes in composition of tears have been reported invarious conditions including not only local eye diseases but systemicconditions such as cancers and diabetes. The dynamic changes incomposition of tears in response to systemic pathophysiologic conditionsrender tears as an alternative source of biomarkers.

The tear film is a kind of hydrated mucin gel containing fluid,electrolytes, proteins and other aqueous components secreted by lacrimalglands and ocular surface epitheoium, and it is covered by a lipid layergenerated by meibomian glands. Tear fluids have been used to assessstress levels in individuals, specific markers such as serotonin,cortisol and DHEA levels are indicative of stress levels. Varioustechniques such as Gas chromatography-mass spectrometry (GC-MS), Liquidchromatography-mass spectrometry (LC-MS), Immunoassays for steroidhormones have been used to measure levels of specific markers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of an aptamer modified for binding to a contactlens surface.

FIG. 2 is a diagram showing binding of a modified aptamer to contactlens surface.

FIG. 3 shows a detection process example of a functionalized contactlens exposed to a biomarker.

DETAILED DESCRIPTION

Despite its non-invasive nature of collection, limited amount of tearfluid compared to other biospecimens such as blood, urine and saliva,tears require a more convenient, standardized method for collectionalong with reliable and reproducible techniques for analysis. Recently,contact lenses have been used for various purposes other than visioncorrection or cosmetics. However, it has been realized that the use ofeyewear devices for other medical purposes, such as interacting withtear fluid are underdeveloped.

One aspect of what is disclosed herein pertains to eyewear devices thatinclude molecules associated therewith that can interact with biomarkerspresent in tears. In one embodiment, disclosed is a contact lens thathas a functionalized surface to enable detection of certain biomarkers.In a more specific embodiment, the contact lens has an aptamerassociated therewith for detecting such biomarker.

Compared to the one-time collection of tears, contact lens enablescumulative detection of biomarker during wearing period and potentiallyincrease sensitivity of the assay. Multiplexing to detect variousbiomarkers at the same time (i.e. via one eyewear device) is achievabledue to the large surface area of contact lens (Contact lens biomarkerschip). According to a certain embodiment, disclosed is an eyewear devicefor detecting at least two different biomarker molecules on eye or intear fluid, wherein the device is configured to rest on the eye andcomprising at least two different biomarker-capture molecules associatedtherewith.

The term “biocapture molecule” refers to a molecule associated with aneyewear device that binds to a target molecule on the eye or present intear fluid. Specific examples of biocapture molecules include antibodiesor aptamers.

The term “aptamer” as used herein pertains to an oligonucleic acid orpeptide molecule that binds to a specific target molecule. When theaptamer includes an oligonucleic acid the oligonucleic acid may besingle or double stranded and may have a fold or other conformationwhere part of the aptamer associates with another part of the aptamer.

Biomarkers that may be targeted by a biocapture molecule associated withan eyewear device may include, but are not limited to, those associatedwith Neurodegenerative diseases (e.g. Parkinson's disease, Alzheimer'sdisease) such as alpha-synuclein, Amyloid Beta, Tau protein, InsulinDegrading Enzyme and amyloid precursor protein. Other biomarkers mayinclude those associated with dry eye such as alpha-1-antitrypsin,calgranulin A, calgranulin B, S100 A4, lactoferrin and lysozyme. Anotherbiomarker may include biomarkers for various cancers, such as but notlimited to, prostate specific antigen, or c-erB-2, VEGF, EGF, and CeA.Other biomarkers may include homocysteine, haemoglobin A1c, glucose, orthyroid hormones. Further still, other biomarkers may includeneurotransmitters and small molecules (dopamine, serotonin, homocysteineetc), inflammatory cytokines (TNF-alpha, IL-1 beta, IL-6, IL-8, IL-12,IFN-gamma, etc), noncytokine proteins (lysozyme, lactoglobin, EGF,lipocalin-1, cystatin S100, alpha1-antitrypsin, alpha-enolase,calgranulin A and B, prolactin inducible protein), MCP-1, lipocalin-1,heat shock protein (HSPs), complement proteins. In a certain embodiment,the biomarker is alpha-1 antichymotrypsin (serpin A3) which has beenshown to be elevated in tears of patients with multiple sclerosis.Salvisberg, C, Proteomics Clin Appl. 2014 April; 8(3-4):185-94. Inanother certain embodiment, the biomarker is directed to TNF-Alpha,which is known to be elevated in diabetic retinopathy and malignantdisease.

Examples of specific aptamers known in the art for various biomarkersinclude, but are not limited to, those described in the following:

-   1. Rahimi, J Vis Exp, 2010 13(39):1955 (amyloid beta)-   2. Wang X, Yang Y, Jia M, et al. The novel amyloid-beta peptide    aptamer inhibits intracellular amyloid-beta peptide toxicity. Neural    Regeneration Research. 2013; 8(1):39-48.    doi:10.3969/j.issn.1673-5374.2013.01.005.-   3. Takahashi et al., Mol Biosyst. 2009 September; 5(9):986-91    (amyloid beta)-   4. Tsukakoshi et al, Biotechnol Lett. 2010 May; 32(5):643-8 (alpha    synuclein)-   5. Krylova et al., Febs Letters, 2005, Volume 579, Issue 6, Pages    1371-1375 (tau)-   6. DE 102010038842 A1 (tau)-   7. Farrar C T, William C M, Hudry E, Hashimoto T, Hyman B T (2014)    RNA Aptamer Probes as Optical Imaging Agents for the Detection of    Amyloid Plaques. PLoS ONE 9(2): e89901-   8. Lee et al., Scientific Reports (Impact Factor: 5.58). 06/2015;    5:10757 (tau)-   9. Sarell et al., J Biol Chem. 2014 Sep. 26; 289(39):26859-71    (Amyloid precursor protein)-   10. US Pat Pub 20130022538 alpha-1-antitrypsin-   11. EP 2316935 A1 (IL-7)

According to another embodiment, disclosed is a method of making afunctionalized eyewear device for detecting a molecule on eye or tearfluid. The method includes attaching an aptamer to a surface of thedevice. The device may be made of a material that provides an availablehydroxyl group (or carboxylic acid, aromatic amine, amide, hydralizide,aldehyde, thiol, or epoxy) for attachment of said aptamer. The methodmay further include silanizing the hydroxyl group. Silanizing thehydroxyl group may involve subjecting the hydroxyl group or other notedgroup to at least one silane compound. Examples of silane compoundsinclude but are not limited to, 3-glycidoxypropyltrimethoxysilane(3-GPTMS), 3-mercaptopropyltrimetoxysilane or3-aminopropyltrimetoxysilane. The method may also involve providing anaptamer that includes a hydrocarbon chain having a terminus with afunctional group that interacts with a terminus of the silane compound.

In certain embodiments, a number of chemical reactions could be used:one end of the aptamer (either 5′- or 3′-end,) can be easily modified byattaching functional groups such as an amine or carboxy (For peptidebased aptamers, these can be modified for attachment or the native aminoor carboxyl termini can be involved for attachment). Then thisfunctional group can be further linked to materials of the contact lenssuch as HEMA (2-hydroxy ethyl methacrylate), silicone hydrogel,methacrylic acid, polyvinyl alcohol (PVA) or N-vinyl pyrrolidone (NVP),polyHEMA or methyl methacrylate (MMA), polyethylene glycol,N,N-dimethylacrylamide, N-carboxyl vinyl ester, or plasma oxidizedsurface via an interactive group such as a hydroxyl that is exposed onthe surface as discussed above. In a further more specific embodiment, alinker can be attached to the surface of contact lens that is then boundto a group on functional group associated with the aptamer.

According to another embodiment, disclosed is a method of detecting abiomarker in tear fluid. The method may involve applying to an eye of asubject a functionalized eyewear device that includes abiomarker-capture molecule associated therewith for a period of time fora biomarker to bind with said biomarker-capture molecule to produce asample-loaded device; and subjecting the sample-loaded device to adetection process. The detection process may involve subjecting thesample-loaded device with chemical reagents that produce a detectablesignal, such as a colorimetric detectable signal. A colorimetricdetectable signal includes, but is not limited to, fluorescence,luminescence, chemiluminescence, or a color formation, or changes in anyof the foregoing, including changes in intensity, duration, anisotropyor polarization, or color change.

In a further embodiment, disclosed is a method of determining a diseasestate that involves applying to an eye of a subject a functionalizedeyewear device that includes a biomarker-capture molecule associatedtherewith for a period of time for a biomarker indicative of the diseasestate to bind with said biomarker-capture molecule to produce asample-loaded device; subjecting said sample-loaded device to adetection process; and determining the disease state based on presenceor amount of said biomarker. The disease state may pertain to, but isnot limited to, a neurological condition, cancer, diabetes, eye disease,or inflammation.

EXAMPLES Example 1: Surface Functionalization with Aptamer for HumanAlpha-Synuclein Oligomer

1. Contact Lens: Claren Ultra-Soo Lens (Interojo Inc.)

Material of this lens is bioxifilcon B with sodium hyaluronate as amajor component. The hydroxyl (—OH) group in sodium hyaluronate is usedfor aptamer attachment.

2. Aptamer:

The 5′-end of aptamer for alpha-synuclein oligomer (T-S0508, publishedin Analytical chemistry 84, 5542-5547) is modified by photocleavableAmino C6 (PCAmC6) to make it bind to silane-modified surface of acontact lens. See FIG. 1.

3. Covalent Attachment of PCAmC6-Aptamer to Contact Lens Surface.

To covalently attach aptamers against target biomarkers to the surfaceof contact lens, two-step chemical reactions was performed:

1) Silanizing the surface of contact lens using3-glycidoxypropyltrimethoxysilane (3-GPTMS): the hydroxyl group ofsodium hyaluronate of contact lens is silanized by 3-GPTMS whose epoxyfunctional group provides stable linkages to modified aptamers. See FIG.2. Alternatively, this step could be achieved by using other chemicalssuch as 3-mercaptopropyltrimetoxysilane or 3-aminopropyltrimetoxysilane,providing mercapto- or amino-functional group, respectively.Procedure:

Incubation of contact lens with 25% 3-GPTMS in deionized H₂O containinga catalytic amount of diisopropylethylamine at 65° C. overnight.Attachment of 3-GPTMS on the surface of contact lens was confirmed byInfrared (IR) spectroscopy.

2) Covalent attachment of PCAmC6-aptamer to the epoxy group of 3-GPTMSon the surface of contact lens: To attach aptamers to the epoxy group of3-GPTMS, aptamers need to be chemically modified to provide a functionalgroup with the capacity to covalently react with the terminal functionalgroup of the silanized surface. PCAmC6 containing amine group withsuitable hydrocarbon spacers (C6) is attached to the 5′end of aptamers.PCAmC6-aptamers can be commercially generated by Gene Link.Procedure:

10 μM (between 5-50 μM) of PCAmC6-aptamers were incubated with 3-GPTMScoated contact lens in 10 mM KOH at 37° C. for 6 hours. Attachment ofPCAmC6-aptamers on the surface of contact lens was confirmed by Infrared(IR) spectroscopy.

Coating the surface of contact lens with PCAmC6-aptamers by performingthe procedure described above was successfully achieved.

Example 2: Detection of Alpha-Synuclein Oligomers Using Contact LensCoated with Aptamers

By wearing the contact lens coated with aptamers, it is expected thatthey can detect potential biomarkers for a range of diseases in the tearfluid. Biomarkers are continuously captured by aptamers during theperiod of wearing contact lens. Captured biomarkers are detected laterusing various already available methods: ELISA,photocleavage-quantitative PCR for aptamer. This greatly improves thedetection threshold for these markers which may only be present in traceamounts and consequently escape detection during one time sampling oftear fluids.

Detection of alpha-synuclein oligomer which is a major component of Lewybodies, pathologic hallmark of Parkinson's disease, was tested using thecontact lens discussed in Example 1. Various biofluids such as blood,cerebrospinal fluid (CSF) and saliva have been tested for detection ofalpha-synuclein as a biomarker for Parkinson's disease. Alpha-synucleinlevels in the CSF have been reported to be correlated with Parkinson'sdisease.

Procedure:

a piece of contact lens coated with aptamers was incubated with variousconcentrations of alpha-synuclein oligomers (0.50.5 μg, 1.0 μg and 1.5μg) in the 96-well plate overnight. After washing three times to removeexcess alpha-synuclein oligomers, contact lens was incubated withprimary antibody against alpha-synuclein overnight. Next day, afterwashing three times, secondary antibody tagged with horse radishperoxidase (HRP) was added and incubated for 1 hour. HRP wasquantitatively measured following DAB reaction. See FIG. 3.

Conclusion

Production of a contact lens functionalized with an aptamer wassuccessfully established. The contact lens coated with aptamer foralpha-synuclein successfully bound to the biomarker and could bedetected. The method can be implemented to attach any of a number ofpossible aptamers to a contact lens for detection.

It should be borne in mind that all patents, patent applications, patentpublications, technical publications, scientific publications, and otherreferences referenced herein are hereby incorporated by reference inthis application in order to more fully describe the state of the art towhich the present invention pertains.

Reference to particular buffers, media, reagents, cells, cultureconditions and the like, or to some subclass of same, is not intended tobe limiting, but should be read to include all such related materialsthat one of ordinary skill in the art would recognize as being ofinterest or value in the particular context in which that discussion ispresented. For example, it is often possible to substitute one buffersystem or culture medium for another, such that a different but knownway is used to achieve the same goals as those to which the use of asuggested method, material or composition is directed.

It is important to an understanding of the present invention to notethat all technical and scientific terms used herein, unless definedherein, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. The techniques employed herein arealso those that are known to one of ordinary skill in the art, unlessstated otherwise. For purposes of more clearly facilitating anunderstanding the invention as disclosed and claimed herein, thefollowing definitions are provided.

While a number of embodiments of the present invention have been shownand described herein in the present context, such embodiments areprovided by way of example only, and not of limitation. Numerousvariations, changes and substitutions will occur to those of skill inthe art without materially departing from the invention herein. Forexample, the present invention need not be limited to best modedisclosed herein, since other applications can equally benefit from theteachings of the present invention. Also, in the claims,means-plus-function and step-plus-function clauses are intended to coverthe structures and acts, respectively, described herein as performingthe recited function and not only structural equivalents or actequivalents, but also equivalent structures or equivalent acts,respectively. Accordingly, all such modifications are intended to beincluded within the scope of this invention as defined in the followingclaims, in accordance with relevant law as to their interpretation.

While one or more embodiments of the present invention have been shownand described herein, such embodiments are provided by way of exampleonly. Variations, changes and substitutions may be made withoutdeparting from the invention herein. Accordingly, it is intended thatthe invention be limited only by the spirit and scope of the appendedclaims. The teachings of all references cited herein are incorporated intheir entirety to the extent not inconsistent with the teachings herein.

The invention claimed is:
 1. A functionalized eyewear device fordetecting a molecule in tear fluid, said device configured to rest onthe eye and comprising a biomarker-capture molecule associated therewiththat binds to alpha-synuclein.
 2. The eyewear device of claim 1, whereinsaid device is a contact lens.
 3. The eyewear device of claim 2, whereincontact lens is comprised of a hydrogel.
 4. The eyewear device of claim3, wherein said hydrogel comprises a hydroxyl group on its surface. 5.The eyewear device of claim 4, wherein said hydroxyl group is reactedwith a silane compound.
 6. The eyewear device of claim 1, wherein saidbiomarker-capture molecule is an aptamer.
 7. The eyewear device of claim6, wherein said aptamer is attached to a hydrocarbon chain having afunctional group that interacts with a terminus of a silane compound. 8.The functionalized eyewear device of claim 6, wherein the aptamer ismodified to comprise a first linker having amino terminus that is boundto a second linker attached to a surface of the eyewear device.
 9. Thefunctionalized eyewear device of claim 8, wherein the aptamer comprisesa 5′ end that is bound to the first linker.
 10. The functionalizedeyewear device of claim 9, wherein the first linker comprises ahydrocarbon chain.
 11. The functionalized eyewear device of claim 6,comprising at least two different aptamers that each bind to a differentbiomarker.
 12. A method of detecting alpha-synuclein in tear fluid, themethod comprising applying to an eye of a subject a functionalizedeyewear device according claim 1 for a period of time foralpha-synuclein in the tear fluid to bind with the biomarker-capturemolecule, wherein alpha-synuclein binds to the biomarker-capturemolecule to produce a sample-loaded device; and subjecting saidsample-loaded device to a detection process.
 13. The method of claim 12,wherein said detection process comprises subjecting said sample-loadeddevice with chemical reagents that produce a colorimetric detectablesignal.